- Email: [email protected]
Mutations in SRCAP, Encoding SNF2-Related CREBBP Activator Protein, Cause Floating-Harbor Syndrome
REPORT Mutations in SRCAP, Encoding SNF2-Related CREBBP Activator Protein, Cause Floating-Harbor Syndrome Rebecca L. Hood,1,2 Matthew A. Lines,3 Sarah M. Nikkel,3,4 Jeremy Schwartzentruber,5 Chandree Beaulieu,6 Ma1gorzata J.M. Nowaczyk,7 Judith Allanson,3 Chong Ae Kim,8 Dagmar Wieczorek,9 Jukka S. Moilanen,10 Didier Lacombe,11 Gabriele Gillessen-Kaesbach,12 Margo L. Whiteford,13 Caio Robledo D.C. Quaio,8 Israel Gomy,8 Debora R. Bertola,8 Beate Albrecht,9 Konrad Platzer,12 George McGillivray,14 Ruobing Zou,2 D. Ross McLeod,15 Albert E. Chudley,16,17 Bernard N. Chodirker,16,17 Janet Marcadier,6 FORGE Canada Consortium,18 Jacek Majewski,5,19 Dennis E. Bulman,2,* Susan M. White,14,20 and Kym M. Boycott3,4,6,* Floating-Harbor syndrome (FHS) is a rare condition characterized by short stature, delayed osseous maturation, expressive-language deficits, and a distinctive facial appearance. Occurrence is generally sporadic, although parent-to-child transmission has been reported on occasion. Employing whole-exome sequencing, we identified heterozygous truncating mutations in SRCAP in five unrelated individuals with sporadic FHS. Sanger sequencing identified mutations in SRCAP in eight more affected persons. Mutations were de novo in all six instances in which parental DNA was available. SRCAP is an SNF2-related chromatin-remodeling factor that serves as a coactivator for CREB-binding protein (CREBBP, better known as CBP, the major cause of Rubinstein-Taybi syndrome [RTS]). Five SRCAP mutations, two of which are recurrent, were identified; all are tightly clustered within a small (111 codon) region of the final exon. These mutations are predicted to abolish three C-terminal AT-hook DNA-binding motifs while leaving the CBP-binding and ATPase domains intact. Our findings show that SRCAP mutations are the major cause of FHS and offer an explanation for the clinical overlap between FHS and RTS.
Floating-Harbor syndrome (FHS [MIM 136140]) is a rare condition characterized by short stature, delayed osseous maturation, language deficits, and a distinctive facial appearance. The dysmorphic features typical of this disorder include a triangular face, short philtrum, wide mouth with a thin vermilion border of the upper lip, and long nose with a narrow bridge, broad base, full tip, and low-hanging columella.1–4 Some degree of intellectual or learning disability is present in most individuals, and language (both receptive and expressive) is typically more severely affected. The name ‘‘Floating Harbor’’ is a portmanteau of Boston Floating Hospital and Harbor General Hospital (Torrance, CA), the two institutions from which the initial case reports originated.1,2 Of the 50 or so cases of FHS in the literature, the majority are sporadic, although four reported instances of parent-tochild transmission suggest that this is an autosomal-dominant disorder in at least some instances.4–7 Some authors have highlighted the clinical overlap between FHS and Rubinstein-Taybi syndrome (RTS [MIM 180849]), which
shares several key features (short stature, a long nose with low-hanging columella, a thin vermilion border of the upper lip, and anomalous thumbs).3,7 Despite the recognition of FHS as a distinct clinical entity more than 25 years ago, no causative mutations have been identified to date. To identify the genetic basis of FHS, we assembled a cohort of 13 unrelated probands, three of whom were previously reported.4 The clinical details of these individuals are presented in Table 1 and Figure 1. To identify FHS-causing mutations, we performed exome capture and high-throughput sequencing of five unrelated affected persons (probands 1–5). Approval of the study design was obtained from the institutional research ethics board (Children’s Hospital of Eastern Ontario), and free and informed consent was obtained from each study subject (or parent, if appropriate) prior to enrollment. We performed exome target enrichment by using the Agilent SureSelect 50 Mb All Exon Kit, and sequencing (Illumina HiSeq) generated 35–40 Gbp of 100 bp paired-end reads
1 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada; 2Ottawa Hospital Research Institute, Ottawa, Ontario K1Y 4E9, Canada; 3Department of Genetics, Children’s Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada; 4Department of Pediatrics, University of Ottawa, Ottawa, Ontario K1H 8L1, Canada; 5McGill University and Genome Quebec Innovation Centre, Montre´al, Que´bec H3A 1A4, Canada; 6Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Ontario K1H 8L1, Canada; 7McMaster University Medical Centre, Hamilton, Ontario L8S 4J9, Canada; 8Unidade de Gene´tica, Instituto da Crianc¸a, Hospital das Clı´nicas-Faculdade de Medicina Universidade de Sa˜o Paulo, Sa˜o Paulo 05403-000, Brazil; 9Institut fu¨r Humangenetik, Universitaetsklinikum Essen, Essen 45147, Germany; 10Department of Clinical Genetics, Oulu University Hospital and University of Oulu, Oulu FI-90029, Finland; 11EA 4576, Laboratoire Maladies Rares: Ge´ne´tique et Me´tabolisme, ¨ r HumangeneService de Ge´ne´tique Me´dicale, Centre Hospitalier Universitaire de Bordeaux, University of Bordeaux, Bordeaux 33076, France; 12Institut fu ¨ beck, Lu ¨ beck 23538, Germany; 13Ferguson-Smith Centre for Clinical Gentetics, Yorkhill Hospital, Glasgow G3 0SJ, Scotland, UK; tik, Universita¨t zu Lu 14 Genetic Health Services Victoria, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Victoria 3052, Australia; 15Department of Medical Genetics, University of Calgary, Calgary, Alberta T3B 6A8, Canada; 16Department of Paediatrics and Child Health, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada; 17Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada; 18FORGE Steering Committee membership is listed in Acknowledgments; 19Department of Human Genetics, McGill University, Montre´al, Que´bec H3A 1B1, Canada; 20Department of Paediatrics, University of Melbourne, Victoria 3010, Australia *Correspondence: [email protected] (K.M.B.), [email protected] (D.E.B.) DOI 10.1016/j.ajhg.2011.12.001. Ó2012 by The American Society of Human Genetics. All rights reserved.
308 The American Journal of Human Genetics 90, 308–313, February 10, 2012
Table 1.
Phenotype of Floating-Harbor Syndrome Probands with SRCAP Mutationsa
The American Journal of Human Genetics 90, 308–313, February 10, 2012 309
Proband
1
2
3
4
5
6
7
8
9
10
11
12
13
Mutation (cDNA)
c.7330C>T
c.7330C>T
c.7330C>T
c.730C>T
c.7549delC
c.7330C>T
c.7330C>T
c.7330C>T
c.7303C>T
c.7303C>T
c.7303C>T
c.7218_ 7219delTC
c.7316dupC
Alteration (protein)
p.Arg2444*
p.Arg2444*
p.Arg2444*
p.Arg2435*
p.Gln2517fs*5 p.Arg2444*
p.Arg2444*
p.Arg2444*
p.Arg2435*
p.Arg2435*
p.Arg2435*
p.Gln2407fs*35 p.Ala2440fs*3
Inheritance
unknown
unknown
unknown
de novo
de novo
de novo
de novo
unknown
unknown
unknown
unknown
de novo
de novo
Sex
M
F
M
M
M
M
M
F
M
M
M
M
M
Ethnicity
French
mixed European
mixed European
Finnish
German and Mexican
Brazilian
German
Caucasian
Caucasian
Brazilian
Brazilian
Chinese
Polish
Paternal age (year)
28
43
29
35
39
32
44
40
34
41
40
40
35
Gestation (weeks)
40
40
38
37
39
40
39
31
41
39
40
40
41
Birth weight (g)
3,040 (0.7 SD)
3,060 (0.6 SD)
2,400 (1.7 SD)
2,620 (0.5 SD)
2,515 (2.2 SD)
2,555 (1.8 SD)
2,430 (2.4 SD)
1,655 (0 SD)
2,900 (1.0 SD)
2,550 (1.8 SD)
2,030 (3.1 SD)
2,800 (1.1 SD)
2,730 (1.5 SD)
Age at diagnosis
3 years
10 years
15 months
11 years
4 years, 3 months
3 years, 3 months
4 years, 4 months
4 years
11 months
7 years, 5 months
8 years
10 years
35 months
ALA
8 years
12 years
12 years
11 years
4 years, 3 months
4 years
4 years, 4 months
10 years, 5 months
11 years
19 years
19 years, 7 months
11 years
7 years, 5 months
Head 54 (þ1 SD) circumference (cm) ALA
0 SD
53 (1 SD)
53.5 (0 SD)
48.5 (1.7 SD)
48 (2 SD)
50 (þ0.7 SD)
49.5 (1 SD)
50.5 (2 SD)
52 (2.5 SD)
56 (0 SD)
53 (0 SD)
51.5 (0.5 SD)
Weight (kg) ALA
25.5 (0 SD)
35.6 (0.8 SD)
N/R
22.4 (3.2 SD)
11 (3.4 SD)
12 (2.5 SD)
12.5 (2.4 SD)
19.3 (3.3 SD)
20 (4.2 SD)
37.6 (5.3 SD)
62.1 (0.7 SD)
35 (0 SD)
20 (1.4 SD)
Height (cm) ALA
123 (0.8 SD)
133.5 (2.2 SD)
134.8 (2.0 SD)
122 (3.1 SD)
86.5 (4.3 SD)
89.8 (3.2 SD)
90 (3.6 SD)
118.5 (3.5 SD)
116.8 (3.9 SD)
145.5 (4.1 SD)
148 (3.8 SD)
139 (0.6 SD)
111 (2.5 SD)
Age at puberty
N/A
12 years
N/A
N/A
N/A
N/A
N/A
N/A
Pubertal age 14 years at CA 11 years
N/R
N/R
10 years
N/A
Prepubertal height
0.8 SD
2.2 SD
2.0 SD
3.1 SD
4.4 SD
3.2 SD
3.6 SD
3.5 SD
N/R
N/R
N/R
3 SD
2.5 SD
BA versus CA
BA 2.5 years at CA 7.5 years
BA 8 years at BA 2 years, CA 11 years 6 months at CA 5 years, 7 months; BA 11 years at CA 9 years, 9 months
BA 2 years at CA 4 years, 8 months; BA 9 years at CA10 years, 8 months
BA 1 year at CA 2 years, 11 months
BA 1 year at BA 3 years at CA 3 years CA 4 years
BA 1 year at CA 5 years
BA 3–6 months at CA 1 year
BA 2 years, 8 months at CA 7 years
BA 3 years at CA 7 years
BA 10 years, 6 months at CA 11 years, 4 months
BA 8 months at CA 2 years, 8 months
Triangular face
þ
þ
þ
þ
þ
N/R
-
þ
þ
þ
þ
þ
þ
Distinctive nose
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Low-hanging columella
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ (Continued on next page)
310 The American Journal of Human Genetics 90, 308–313, February 10, 2012
Table 1.
Continued
Proband
1
2
3
4
5
6
7
8
9
10
11
12
13
Short philtrum
þ
þ
þ
þ
þ
þ
þ
þ
þ
-
þ
-
þ
Thin upper vermilion border
þ
þ
þ
þ
-
-
þ
þ
þ
-
-
þ
þ
Wide mouth
þ
-
þ
þ
þ/
þ
þ
þ
þ
þ
þ
þ
þ
Low-set ears
þ
þ
-
þ
þ
-
þ
þ
þ
-
-
þ
þ
Broad thumbs -
þ
-
þ
þ
þ
-
N/R
N/R
N/R
þ
þ
þ
Broad fingertips
-
þ
þ
þ
N/R
N/R
-
N/R
þ
N/R
þ
þ
þ
Brachydactyly -
þ
þ
þ
-
fifth toes
-
þ
-
N/R
þ
þ
þ
Clinodactyly
-
N/R
radial deviation þ fifth distal phalanx
þ
-
-
þ
þ
N/R
þ
þ
þ
Other skeletal
N/R
N/R
dislocated radial head, 11 rib pairs
N/R
short fifth metacarpal
N/R
clavicular hypoplasia
kyphoscoliosis
dysplastic hips
11 rib pairs, ivory epiphyses in distal phalanges
short middle phalanges of second and fifth fingers
short first metacarpal
short fifth metacarpal
Dental issues
N/R
N/R
maxillary retrusion, underbite
caries, microdontia
N/R
N/R
N/R
caries, delayed loss of primary teeth
N/R
N/R
normal
caries, microdontia, underbite
N/R
Other health issues
hypospadias, hydroceliac disease nephrosis, nephrocalcinosis, recurrent otitis media
aortic coarctation (mild)
cryptorchidism, hyperopia hyperopia, conductive hearing loss
ASD, hyperopia, unilateral renal pelviectasis
mesocardia, constipation persistent left superior vena cava, conductive hearing loss
bilateral epididymal cysts, left varicocele
unilateral cleft lip, cryptorchidism
Intellectual development
borderline normal
normal
borderline normal
borderline normal
normal
moderate delay
borderline normal
mild intellectual moderately disability severe learning disability
significant intellectual disability
intellectual disability
borderline normal
mild intellectual disability
Expressive language delay
delay
moderate delay
moderate delay
impairment
borderline normal, bilingual
moderate delay
moderate delay
severe delay
moderate delay
some words
moderate delay
moderate delay
moderate delay
Education
mainstream mainstream with support with support
mainstream with support
special school
mainstream
N/A
mainstream with support
modified classroom
special school
special school
special school modified classroom
mainstream with support
Microarray findings and (type)
normal (44K)
normal (180K Agilent)
normal (105K)
7q31dup (paternally inherited)
Xp22.31dup N/A (maternally inherited)
N/A
N/A
N/A
normal 22q11 normal (Agilent 6.1)
normal (244K Agilent)
Reference
White et al,4 White et al,4 person 10 person 9
this report
this report
this report
this report
this report
White et al,4 person 8
this report
this report
this report
normal (Affymetrix 2.7M)
this report
bilateral posterior strabismus inguinal hernia, urethral valves, cryptorchidism, umbilical hernia VPI, hearing loss
this report
Numbering of mutations is relative to NM_006662.2 (gene) and NP_006653 (protein). Abbreviations are as follows: ALA, at last assessment; N/A, Not Applicable; N/R, Not Reported; þ, Feature Present; -, Feature Absent; SD, standard deviations; BA, bone age; CA, chronological age; ASD, atrial septal defect; and VPI, velopharyngeal incompetence. a This table summarizes the clinical findings in all study participants. Five participants (discovery cohort; individuals 1–5) underwent exome sequencing. Mutations in individuals 6–13 (validation cohort) were identified with Sanger sequencing.
Figure 1. Floating-Harbor Syndrome Due to SRCAP Mutations Clinical photos depicting 9 of 13 unrelated FHS probands with a confirmed SRCAP mutation are shown with the characteristically triangular face, long eyelashes, typical nose (long and narrow nasal bridge, broad base, full tip, and low-hanging columella), short philtrum, wide mouth, thin vermilion border of the upper lip, short chin, and low-set, posteriorly rotated ears. Clinical details concerning all study participants are presented in Table 1. (A) Individual 1. Age 3 years; age 8 years. (B) Individual 3. Age 3 years, 7 months; age 11 years, 5 months. (C) Individual 4. Age 4 years, 6 months. (D) Individual 8. Age 4 years; age 10 years, 5 months. (E) Individual 9. Age 11 years. (F) Individual 10. Age 15 months; age 19 years. (G) Individual 11. Age 19 years, 7 months. (H) Individual 12. Age 4 years. (I) Individual 13. Age 3 years; age 7 years, 5 months.
per sample. Reads were preprocessed (trimmed) and aligned to hg19 (see Web Resources for list of tools). We used an inhouse annotation pipeline to identify coding and splicesite variants that met a minimum quality threshold (i.e., R20% of reads supported the variant). Next, we filtered the variants to exclude common polymorphisms (>1% minor-allele frequency) represented in dbSNP131, in the 1000 Genomes pilot release, or in 270 exomes sequenced for individuals with unrelated disorders at our center. Presuming FHS to be an autosomal-dominant condition, we identified genes containing a single rare variant in each of several probands in a combinatorial fashion. Table 2 lists the numbers of potential candidate genes containing rare variants in any n probands as n is increased. Of five sequenced individuals with classic FHS, we noted that all contained heterozygous truncating variants clustered in the final (34th) exon of a single gene, SRCAP (encoding SNF2-related CREBBP activator protein). To confirm SRCAP as the gene mutated in FHS, we identified SRCAP exon 34 mutations with Sanger sequencing in a validation cohort of eight more unrelated probands (Table 1 and Figure 2; Figure S1, available online). All of these mutations are truncating (nonsense or frameshift) alleles, tightly clustered between codons 2,407 and 2,517; none are represented in dbSNP131, 1000 Genomes, or the National Heart, Lung,
and Blood Institute (NHLBI) Exome Variant Server (see Web Resources). Two mutations in particular, c.7330C>T (NM_006662.2) (p.Arg2444* [NP_006653]) in six individuals and c.7303C>T (p.Arg2435*) in four individuals, accounted for the large majority of mutations. FHS-causing mutations were shown to be de novo in all six instances in which parental DNA samples were available. SRCAP encodes a switch/sucrose nonfermentable (SWI/ SNF)-type chromatin-remodeling ATPase identified in a two-hybrid screen for interacting partners of CREBbinding protein (CREBBP, hereafter called CBP).8 In reporter assays, SRCAP is a potent coactivator for CREB and CBP-mediated transcription.8,9 In transgenic Drosophila, exogenous SRCAP colocalizes with transcriptionally active chromatin and augments CBP’s presence at these sites.10
Table 2.
Variant Analysis in Floating-Harbor Syndrome Probands
Any X of five individuals
4
5
Number of genes containing missense, 2,375 307 48 8 nonsense, insertion, deletion, or splice-site variants
2
Allele frequency %1% in dbSNP131 and 1000 Genomes; not represented in 270 local exomes
1
1,178
2
70
3
3 SRCAP SRCAP
The American Journal of Human Genetics 90, 308–313, February 10, 2012 311
Figure 2. Locations of FHS-Causing Mutations within SRCAP (A) Intron-exon structure of SRCAP. Exon 34 mutation cluster is indicated by a red bar. (B) Domain architecture of SRCAP8–10 indicates amino acid positions of recognized domains and FHS-causing mutations. All probands are heterozygous for truncating mutations at the positions shown. The ATPase domain of SRCAP is divided into two sections, one containing conserved motifs I-IV and one containing V-VI. The following abbreviation is used: HSA, Helicase-SANT-associated domain.
Affinity-purified SRCAP precipitates as a large complex that catalyzes ATP-dependent substitution of the variant histone H2A.Z into nucleosomes.11 This activity has been confirmed by knockdown experiments with natural promoters, and it is correlated with in vivo target-gene expression.12 Separately, SRCAP has also been shown to transduce signals belonging to the nuclear (steroid) hormone receptor and Notch pathways, indicating that it has diverse roles in gene expression.10,13 In keeping with its multiple coactivator roles, SRCAP contains several discrete functional domains.8–10 These domains include an SNF2-like ATPase, an N-terminal HSA (Helicase-SANT-associated) domain, and three C-terminal AT-hook DNA-binding motifs; the CBP interaction domain of SRCAP is located centrally (Figure 2). Given the structure of SRCAP, the nonrandom clustering of truncating mutations seen in our study participants is strongly suggestive of a dominant-negative disease mechanism due to loss of one or more critical domain(s), for instance the three C-terminal AT-hook motifs. Several more arguments support this. First, in reporter assays, the major transactivation function of SRCAP is located in a 655 residue C-terminal fragment abolished by FHS-causing truncations.9 Second, expression of a construct solely consisting of the CBP interaction domain of SRCAP strongly inhibits CREB-mediated transactivation in a dominant-negative fashion.9 Third, the Database of Genomic Variants (see Web Resources) contains two HapMap control individuals who bear a 208 kb deletion copy-number variation (#2,209) containing SRCAP and nine adjacent genes and who have no reported phenotype. In general, the phenotype of persons with SRCAP mutations is concordant with earlier clinical descriptions of FHS (Table 1 and Figure 1), and nearly all individuals have short stature and expressive-language impairment. Despite the remarkable similarity among mutations seen in our study subjects, cognitive outcomes ranging from ‘‘normal’’ to ‘‘significant intellectual disability’’ were reported. It is
unclear whether genetic modifier(s) and/or currently unidentified environmental factors are responsible. Many of our study subjects had additional systemic malformations, particularly genitourinary (eight individuals) and cardiac (three individuals) malformations. Again, genotype-phenotype correlations explaining these features are lacking. Given that FHS is a dominant condition exhibiting a high de novo mutation rate, one might expect a paternal age effect to be present, and indeed the mean paternal age of the affected individuals was advanced (36.9 years; range: 29–44 years). Importantly, our findings suggest a basis for the longrecognized phenotypic overlap between FHS and RTS, the latter of which is caused by alterations in CBP or its homolog, p300.14–16 Because alterations in both CBP and SRCAP are expected to produce widespread target-gene dysregulation, further studies are needed before we can determine which transcriptional targets are primarily responsible for each phenotype and whether any of these might be valid therapeutic targets. The availability of a molecular test for FHS will greatly improve the reliability of a formerly challenging clinical diagnosis. Historically, a diagnosis of FHS has relied upon the presence of typical facial features because many other key findings (e.g., short stature and language impairment) are nonspecific. The advent of molecular diagnosis for this condition will allow us to gain a better appreciation of the true prevalence and phenotypic spectrum of FHS. Supplemental Data Supplemental Data include one figure and can be found with this article online at http://www.cell.com/AJHG/.
Acknowledgments The authors would first like to thank the study participants and their families, without whose participation and cooperation this work would not have been possible. This work was funded
312 The American Journal of Human Genetics 90, 308–313, February 10, 2012
by the government of Canada through Genome Canada, the Canadian Institutes of Health Research (CIHR), and the Ontario Genomics Institute (OGI-049). Additional funding was provided by Genome Que´bec and Genome British Columbia. K.M.B. is supported by a Clinical Investigatorship Award from the CIHR Institute of Genetics. This work was selected for study by the FORGE Canada Steering Committee, consisting of K. Boycott (University of Ottawa), J. Friedman (University of British Columbia), J. Michaud (University of Montreal), F. Bernier (University of Calgary), M. Brudno (University of Toronto), B. Fernandez (Memorial University), B. Knoppers (McGill University), M. Samuels (Universite´ de Montreal), and S. Scherer (University of Toronto). Received: November 22, 2011 Revised: December 5, 2011 Accepted: December 7, 2011 Published online: January 19, 2012
5.
6.
7.
8.
Web Resources
9.
The URLs for data presented herein are as follows: Database of Genomic Variants, http://projects.tcag.ca/variation/ FASTX-Toolkit, http://hannonlab.cshl.edu/fastx_toolkit/ NHLBI Exome Variant Server, http://evs.gs.washington.edu/EVS/ Online Mendelian Inheritance in Man (OMIM), http://www. omim.org Picard, http://picard.sourceforge.net/ SAMtools, http://samtools.sourceforge.net/
10.
11.
Accession Numbers The NCBI accession number for the SRCAP sequence reported in this paper is NM_006662.2 and local identifiers are as follows:
NM_006662.2: c.7330C>T (NCBI ss477606270) NM_006662.2: c.7303C>T (NCBI ss477606271) NM_006662.2: c.7549delC (NCBI ss477606272) NM_006662.2: c.7218_7219delTC (NCBI ss477606273) NM_006662.2: c.7316dupC (NCBI ss477606274)
12.
13.
14.
References 1. Pelletier, G., and Feingold, M. (1973). Case report 1. In Syndrome Identification, D. Bergsma, ed. (White Plains, NY: National Foundation-March of Dimes), pp. 8–9. 2. Leisti, J., Hollister, D.W., and Rimoin, D.L. (1975). The FloatingHarbor syndrome. Birth Defects Orig. Artic. Ser. 11, 305. 3. Robinson, P.L., Shohat, M., Winter, R.M., Conte, W.J., Gordon-Nesbitt, D., Feingold, M., Laron, Z., and Rimoin, D.L. (1988). A unique association of short stature, dysmorphic features, and speech impairment (Floating-Harbor syndrome). J. Pediatr. 113, 703–706. 4. White, S.M., Morgan, A., Da Costa, A., Lacombe, D., Knight, S.J., Houlston, R., Whiteford, M.L., Newbury-Ecob,
15.
16.
R.A., and Hurst, J.A. (2010). The phenotype of FloatingHarbor syndrome in 10 patients. Am. J. Med. Genet. A. 152A, 821–829. Lacombe, D., Patton, M.A., Elleau, C., and Battin, J. (1995). Floating-Harbor syndrome: Description of a further patient, review of the literature, and suggestion of autosomal dominant inheritance. Eur. J. Pediatr. 154, 658–661. ˜aloza, J.M., Garcı´a-Cruz, D., Da´valos, I.P., Da´valos, N.O., Pen Garcı´a-Cruz, M.O., Pe´rez-Rulfo, D., and Sa´nchez-Corona, J. (2003). A variant example of familial Floating-Harbor syndrome? Genet. Couns. 14, 31–37. Arpin, S., Afenjar, A., Dubern, B., Toutain, A., Cabrol, S., and He´ron, D. (2012). Floating-Harbor Syndrome: Report on a case in a mother and daughter, further evidence of autosomal dominant inheritance. Clin. Dysmorphol. 21, 11–14. Johnston, H., Kneer, J., Chackalaparampil, I., Yaciuk, P., and Chrivia, J. (1999). Identification of a novel SNF2/SWI2 protein family member, SRCAP, which interacts with CREB-binding protein. J. Biol. Chem. 274, 16370–16376. Monroy, M.A., Ruhl, D.D., Xu, X., Granner, D.K., Yaciuk, P., and Chrivia, J.C. (2001). Regulation of cAMP-responsive elementbinding protein-mediated transcription by the SNF2/SWIrelated protein, SRCAP. J. Biol. Chem. 276, 40721–40726. Eissenberg, J.C., Wong, M., and Chrivia, J.C. (2005). Human SRCAP and Drosophila melanogaster DOM are homologs that function in the notch signaling pathway. Mol. Cell. Biol. 25, 6559–6569. Ruhl, D.D., Jin, J., Cai, Y., Swanson, S., Florens, L., Washburn, M.P., Conaway, R.C., Conaway, J.W., and Chrivia, J.C. (2006). Purification of a human SRCAP complex that remodels chromatin by incorporating the histone variant H2A.Z into nucleosomes. Biochemistry 45, 5671–5677. Wong, M.M., Cox, L.K., and Chrivia, J.C. (2007). The chromatin remodeling protein, SRCAP, is critical for deposition of the histone variant H2A.Z at promoters. J. Biol. Chem. 282, 26132–26139. Monroy, M.A., Schott, N.M., Cox, L., Chen, J.D., Ruh, M., and Chrivia, J.C. (2003). SNF2-related CBP activator protein (SRCAP) functions as a coactivator of steroid receptormediated transcription through synergistic interactions with CARM-1 and GRIP-1. Mol. Endocrinol. 17, 2519–2528. Petrij, F., Giles, R.H., Dauwerse, H.G., Saris, J.J., Hennekam, R.C., Masuno, M., Tommerup, N., van Ommen, G.J., Goodman, R.H., Peters, D.J., et al. (1995). Rubinstein-Taybi syndrome caused by mutations in the transcriptional co-activator CBP. Nature 376, 348–351. ¨ rek, Y., Bartholdi, D., Roelfsema, J.H., White, S.J., Ariyu Niedrist, D., Papadia, F., Bacino, C.A., den Dunnen, J.T., van Ommen, G.J., Breuning, M.H., et al. (2005). Genetic heterogeneity in Rubinstein-Taybi syndrome: Mutations in both the CBP and EP300 genes cause disease. Am. J. Hum. Genet. 76, 572–580. Stevens, C.A. (2002). Rubinstein-Taybi Syndrome. In GeneReviews, R.A. Pagon, ed. (Seattle: University of Washington), NCBI bookshelf ID: NBK1526.
The American Journal of Human Genetics 90, 308–313, February 10, 2012 313
Floating-Harbor syndrome (FHS [MIM 136140]) is a rare condition characterized by short stature, delayed osseous maturation, language deficits, and a distinctive facial appearance. The dysmorphic features typical of this disorder include a triangular face, short philtrum, wide mouth with a thin vermilion border of the upper lip, and long nose with a narrow bridge, broad base, full tip, and low-hanging columella.1–4 Some degree of intellectual or learning disability is present in most individuals, and language (both receptive and expressive) is typically more severely affected. The name ‘‘Floating Harbor’’ is a portmanteau of Boston Floating Hospital and Harbor General Hospital (Torrance, CA), the two institutions from which the initial case reports originated.1,2 Of the 50 or so cases of FHS in the literature, the majority are sporadic, although four reported instances of parent-tochild transmission suggest that this is an autosomal-dominant disorder in at least some instances.4–7 Some authors have highlighted the clinical overlap between FHS and Rubinstein-Taybi syndrome (RTS [MIM 180849]), which
shares several key features (short stature, a long nose with low-hanging columella, a thin vermilion border of the upper lip, and anomalous thumbs).3,7 Despite the recognition of FHS as a distinct clinical entity more than 25 years ago, no causative mutations have been identified to date. To identify the genetic basis of FHS, we assembled a cohort of 13 unrelated probands, three of whom were previously reported.4 The clinical details of these individuals are presented in Table 1 and Figure 1. To identify FHS-causing mutations, we performed exome capture and high-throughput sequencing of five unrelated affected persons (probands 1–5). Approval of the study design was obtained from the institutional research ethics board (Children’s Hospital of Eastern Ontario), and free and informed consent was obtained from each study subject (or parent, if appropriate) prior to enrollment. We performed exome target enrichment by using the Agilent SureSelect 50 Mb All Exon Kit, and sequencing (Illumina HiSeq) generated 35–40 Gbp of 100 bp paired-end reads
1 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada; 2Ottawa Hospital Research Institute, Ottawa, Ontario K1Y 4E9, Canada; 3Department of Genetics, Children’s Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada; 4Department of Pediatrics, University of Ottawa, Ottawa, Ontario K1H 8L1, Canada; 5McGill University and Genome Quebec Innovation Centre, Montre´al, Que´bec H3A 1A4, Canada; 6Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Ontario K1H 8L1, Canada; 7McMaster University Medical Centre, Hamilton, Ontario L8S 4J9, Canada; 8Unidade de Gene´tica, Instituto da Crianc¸a, Hospital das Clı´nicas-Faculdade de Medicina Universidade de Sa˜o Paulo, Sa˜o Paulo 05403-000, Brazil; 9Institut fu¨r Humangenetik, Universitaetsklinikum Essen, Essen 45147, Germany; 10Department of Clinical Genetics, Oulu University Hospital and University of Oulu, Oulu FI-90029, Finland; 11EA 4576, Laboratoire Maladies Rares: Ge´ne´tique et Me´tabolisme, ¨ r HumangeneService de Ge´ne´tique Me´dicale, Centre Hospitalier Universitaire de Bordeaux, University of Bordeaux, Bordeaux 33076, France; 12Institut fu ¨ beck, Lu ¨ beck 23538, Germany; 13Ferguson-Smith Centre for Clinical Gentetics, Yorkhill Hospital, Glasgow G3 0SJ, Scotland, UK; tik, Universita¨t zu Lu 14 Genetic Health Services Victoria, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Victoria 3052, Australia; 15Department of Medical Genetics, University of Calgary, Calgary, Alberta T3B 6A8, Canada; 16Department of Paediatrics and Child Health, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada; 17Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada; 18FORGE Steering Committee membership is listed in Acknowledgments; 19Department of Human Genetics, McGill University, Montre´al, Que´bec H3A 1B1, Canada; 20Department of Paediatrics, University of Melbourne, Victoria 3010, Australia *Correspondence: [email protected] (K.M.B.), [email protected] (D.E.B.) DOI 10.1016/j.ajhg.2011.12.001. Ó2012 by The American Society of Human Genetics. All rights reserved.
308 The American Journal of Human Genetics 90, 308–313, February 10, 2012
Table 1.
Phenotype of Floating-Harbor Syndrome Probands with SRCAP Mutationsa
The American Journal of Human Genetics 90, 308–313, February 10, 2012 309
Proband
1
2
3
4
5
6
7
8
9
10
11
12
13
Mutation (cDNA)
c.7330C>T
c.7330C>T
c.7330C>T
c.730C>T
c.7549delC
c.7330C>T
c.7330C>T
c.7330C>T
c.7303C>T
c.7303C>T
c.7303C>T
c.7218_ 7219delTC
c.7316dupC
Alteration (protein)
p.Arg2444*
p.Arg2444*
p.Arg2444*
p.Arg2435*
p.Gln2517fs*5 p.Arg2444*
p.Arg2444*
p.Arg2444*
p.Arg2435*
p.Arg2435*
p.Arg2435*
p.Gln2407fs*35 p.Ala2440fs*3
Inheritance
unknown
unknown
unknown
de novo
de novo
de novo
de novo
unknown
unknown
unknown
unknown
de novo
de novo
Sex
M
F
M
M
M
M
M
F
M
M
M
M
M
Ethnicity
French
mixed European
mixed European
Finnish
German and Mexican
Brazilian
German
Caucasian
Caucasian
Brazilian
Brazilian
Chinese
Polish
Paternal age (year)
28
43
29
35
39
32
44
40
34
41
40
40
35
Gestation (weeks)
40
40
38
37
39
40
39
31
41
39
40
40
41
Birth weight (g)
3,040 (0.7 SD)
3,060 (0.6 SD)
2,400 (1.7 SD)
2,620 (0.5 SD)
2,515 (2.2 SD)
2,555 (1.8 SD)
2,430 (2.4 SD)
1,655 (0 SD)
2,900 (1.0 SD)
2,550 (1.8 SD)
2,030 (3.1 SD)
2,800 (1.1 SD)
2,730 (1.5 SD)
Age at diagnosis
3 years
10 years
15 months
11 years
4 years, 3 months
3 years, 3 months
4 years, 4 months
4 years
11 months
7 years, 5 months
8 years
10 years
35 months
ALA
8 years
12 years
12 years
11 years
4 years, 3 months
4 years
4 years, 4 months
10 years, 5 months
11 years
19 years
19 years, 7 months
11 years
7 years, 5 months
Head 54 (þ1 SD) circumference (cm) ALA
0 SD
53 (1 SD)
53.5 (0 SD)
48.5 (1.7 SD)
48 (2 SD)
50 (þ0.7 SD)
49.5 (1 SD)
50.5 (2 SD)
52 (2.5 SD)
56 (0 SD)
53 (0 SD)
51.5 (0.5 SD)
Weight (kg) ALA
25.5 (0 SD)
35.6 (0.8 SD)
N/R
22.4 (3.2 SD)
11 (3.4 SD)
12 (2.5 SD)
12.5 (2.4 SD)
19.3 (3.3 SD)
20 (4.2 SD)
37.6 (5.3 SD)
62.1 (0.7 SD)
35 (0 SD)
20 (1.4 SD)
Height (cm) ALA
123 (0.8 SD)
133.5 (2.2 SD)
134.8 (2.0 SD)
122 (3.1 SD)
86.5 (4.3 SD)
89.8 (3.2 SD)
90 (3.6 SD)
118.5 (3.5 SD)
116.8 (3.9 SD)
145.5 (4.1 SD)
148 (3.8 SD)
139 (0.6 SD)
111 (2.5 SD)
Age at puberty
N/A
12 years
N/A
N/A
N/A
N/A
N/A
N/A
Pubertal age 14 years at CA 11 years
N/R
N/R
10 years
N/A
Prepubertal height
0.8 SD
2.2 SD
2.0 SD
3.1 SD
4.4 SD
3.2 SD
3.6 SD
3.5 SD
N/R
N/R
N/R
3 SD
2.5 SD
BA versus CA
BA 2.5 years at CA 7.5 years
BA 8 years at BA 2 years, CA 11 years 6 months at CA 5 years, 7 months; BA 11 years at CA 9 years, 9 months
BA 2 years at CA 4 years, 8 months; BA 9 years at CA10 years, 8 months
BA 1 year at CA 2 years, 11 months
BA 1 year at BA 3 years at CA 3 years CA 4 years
BA 1 year at CA 5 years
BA 3–6 months at CA 1 year
BA 2 years, 8 months at CA 7 years
BA 3 years at CA 7 years
BA 10 years, 6 months at CA 11 years, 4 months
BA 8 months at CA 2 years, 8 months
Triangular face
þ
þ
þ
þ
þ
N/R
-
þ
þ
þ
þ
þ
þ
Distinctive nose
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Low-hanging columella
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ (Continued on next page)
310 The American Journal of Human Genetics 90, 308–313, February 10, 2012
Table 1.
Continued
Proband
1
2
3
4
5
6
7
8
9
10
11
12
13
Short philtrum
þ
þ
þ
þ
þ
þ
þ
þ
þ
-
þ
-
þ
Thin upper vermilion border
þ
þ
þ
þ
-
-
þ
þ
þ
-
-
þ
þ
Wide mouth
þ
-
þ
þ
þ/
þ
þ
þ
þ
þ
þ
þ
þ
Low-set ears
þ
þ
-
þ
þ
-
þ
þ
þ
-
-
þ
þ
Broad thumbs -
þ
-
þ
þ
þ
-
N/R
N/R
N/R
þ
þ
þ
Broad fingertips
-
þ
þ
þ
N/R
N/R
-
N/R
þ
N/R
þ
þ
þ
Brachydactyly -
þ
þ
þ
-
fifth toes
-
þ
-
N/R
þ
þ
þ
Clinodactyly
-
N/R
radial deviation þ fifth distal phalanx
þ
-
-
þ
þ
N/R
þ
þ
þ
Other skeletal
N/R
N/R
dislocated radial head, 11 rib pairs
N/R
short fifth metacarpal
N/R
clavicular hypoplasia
kyphoscoliosis
dysplastic hips
11 rib pairs, ivory epiphyses in distal phalanges
short middle phalanges of second and fifth fingers
short first metacarpal
short fifth metacarpal
Dental issues
N/R
N/R
maxillary retrusion, underbite
caries, microdontia
N/R
N/R
N/R
caries, delayed loss of primary teeth
N/R
N/R
normal
caries, microdontia, underbite
N/R
Other health issues
hypospadias, hydroceliac disease nephrosis, nephrocalcinosis, recurrent otitis media
aortic coarctation (mild)
cryptorchidism, hyperopia hyperopia, conductive hearing loss
ASD, hyperopia, unilateral renal pelviectasis
mesocardia, constipation persistent left superior vena cava, conductive hearing loss
bilateral epididymal cysts, left varicocele
unilateral cleft lip, cryptorchidism
Intellectual development
borderline normal
normal
borderline normal
borderline normal
normal
moderate delay
borderline normal
mild intellectual moderately disability severe learning disability
significant intellectual disability
intellectual disability
borderline normal
mild intellectual disability
Expressive language delay
delay
moderate delay
moderate delay
impairment
borderline normal, bilingual
moderate delay
moderate delay
severe delay
moderate delay
some words
moderate delay
moderate delay
moderate delay
Education
mainstream mainstream with support with support
mainstream with support
special school
mainstream
N/A
mainstream with support
modified classroom
special school
special school
special school modified classroom
mainstream with support
Microarray findings and (type)
normal (44K)
normal (180K Agilent)
normal (105K)
7q31dup (paternally inherited)
Xp22.31dup N/A (maternally inherited)
N/A
N/A
N/A
normal 22q11 normal (Agilent 6.1)
normal (244K Agilent)
Reference
White et al,4 White et al,4 person 10 person 9
this report
this report
this report
this report
this report
White et al,4 person 8
this report
this report
this report
normal (Affymetrix 2.7M)
this report
bilateral posterior strabismus inguinal hernia, urethral valves, cryptorchidism, umbilical hernia VPI, hearing loss
this report
Numbering of mutations is relative to NM_006662.2 (gene) and NP_006653 (protein). Abbreviations are as follows: ALA, at last assessment; N/A, Not Applicable; N/R, Not Reported; þ, Feature Present; -, Feature Absent; SD, standard deviations; BA, bone age; CA, chronological age; ASD, atrial septal defect; and VPI, velopharyngeal incompetence. a This table summarizes the clinical findings in all study participants. Five participants (discovery cohort; individuals 1–5) underwent exome sequencing. Mutations in individuals 6–13 (validation cohort) were identified with Sanger sequencing.
Figure 1. Floating-Harbor Syndrome Due to SRCAP Mutations Clinical photos depicting 9 of 13 unrelated FHS probands with a confirmed SRCAP mutation are shown with the characteristically triangular face, long eyelashes, typical nose (long and narrow nasal bridge, broad base, full tip, and low-hanging columella), short philtrum, wide mouth, thin vermilion border of the upper lip, short chin, and low-set, posteriorly rotated ears. Clinical details concerning all study participants are presented in Table 1. (A) Individual 1. Age 3 years; age 8 years. (B) Individual 3. Age 3 years, 7 months; age 11 years, 5 months. (C) Individual 4. Age 4 years, 6 months. (D) Individual 8. Age 4 years; age 10 years, 5 months. (E) Individual 9. Age 11 years. (F) Individual 10. Age 15 months; age 19 years. (G) Individual 11. Age 19 years, 7 months. (H) Individual 12. Age 4 years. (I) Individual 13. Age 3 years; age 7 years, 5 months.
per sample. Reads were preprocessed (trimmed) and aligned to hg19 (see Web Resources for list of tools). We used an inhouse annotation pipeline to identify coding and splicesite variants that met a minimum quality threshold (i.e., R20% of reads supported the variant). Next, we filtered the variants to exclude common polymorphisms (>1% minor-allele frequency) represented in dbSNP131, in the 1000 Genomes pilot release, or in 270 exomes sequenced for individuals with unrelated disorders at our center. Presuming FHS to be an autosomal-dominant condition, we identified genes containing a single rare variant in each of several probands in a combinatorial fashion. Table 2 lists the numbers of potential candidate genes containing rare variants in any n probands as n is increased. Of five sequenced individuals with classic FHS, we noted that all contained heterozygous truncating variants clustered in the final (34th) exon of a single gene, SRCAP (encoding SNF2-related CREBBP activator protein). To confirm SRCAP as the gene mutated in FHS, we identified SRCAP exon 34 mutations with Sanger sequencing in a validation cohort of eight more unrelated probands (Table 1 and Figure 2; Figure S1, available online). All of these mutations are truncating (nonsense or frameshift) alleles, tightly clustered between codons 2,407 and 2,517; none are represented in dbSNP131, 1000 Genomes, or the National Heart, Lung,
and Blood Institute (NHLBI) Exome Variant Server (see Web Resources). Two mutations in particular, c.7330C>T (NM_006662.2) (p.Arg2444* [NP_006653]) in six individuals and c.7303C>T (p.Arg2435*) in four individuals, accounted for the large majority of mutations. FHS-causing mutations were shown to be de novo in all six instances in which parental DNA samples were available. SRCAP encodes a switch/sucrose nonfermentable (SWI/ SNF)-type chromatin-remodeling ATPase identified in a two-hybrid screen for interacting partners of CREBbinding protein (CREBBP, hereafter called CBP).8 In reporter assays, SRCAP is a potent coactivator for CREB and CBP-mediated transcription.8,9 In transgenic Drosophila, exogenous SRCAP colocalizes with transcriptionally active chromatin and augments CBP’s presence at these sites.10
Table 2.
Variant Analysis in Floating-Harbor Syndrome Probands
Any X of five individuals
4
5
Number of genes containing missense, 2,375 307 48 8 nonsense, insertion, deletion, or splice-site variants
2
Allele frequency %1% in dbSNP131 and 1000 Genomes; not represented in 270 local exomes
1
1,178
2
70
3
3 SRCAP SRCAP
The American Journal of Human Genetics 90, 308–313, February 10, 2012 311
Figure 2. Locations of FHS-Causing Mutations within SRCAP (A) Intron-exon structure of SRCAP. Exon 34 mutation cluster is indicated by a red bar. (B) Domain architecture of SRCAP8–10 indicates amino acid positions of recognized domains and FHS-causing mutations. All probands are heterozygous for truncating mutations at the positions shown. The ATPase domain of SRCAP is divided into two sections, one containing conserved motifs I-IV and one containing V-VI. The following abbreviation is used: HSA, Helicase-SANT-associated domain.
Affinity-purified SRCAP precipitates as a large complex that catalyzes ATP-dependent substitution of the variant histone H2A.Z into nucleosomes.11 This activity has been confirmed by knockdown experiments with natural promoters, and it is correlated with in vivo target-gene expression.12 Separately, SRCAP has also been shown to transduce signals belonging to the nuclear (steroid) hormone receptor and Notch pathways, indicating that it has diverse roles in gene expression.10,13 In keeping with its multiple coactivator roles, SRCAP contains several discrete functional domains.8–10 These domains include an SNF2-like ATPase, an N-terminal HSA (Helicase-SANT-associated) domain, and three C-terminal AT-hook DNA-binding motifs; the CBP interaction domain of SRCAP is located centrally (Figure 2). Given the structure of SRCAP, the nonrandom clustering of truncating mutations seen in our study participants is strongly suggestive of a dominant-negative disease mechanism due to loss of one or more critical domain(s), for instance the three C-terminal AT-hook motifs. Several more arguments support this. First, in reporter assays, the major transactivation function of SRCAP is located in a 655 residue C-terminal fragment abolished by FHS-causing truncations.9 Second, expression of a construct solely consisting of the CBP interaction domain of SRCAP strongly inhibits CREB-mediated transactivation in a dominant-negative fashion.9 Third, the Database of Genomic Variants (see Web Resources) contains two HapMap control individuals who bear a 208 kb deletion copy-number variation (#2,209) containing SRCAP and nine adjacent genes and who have no reported phenotype. In general, the phenotype of persons with SRCAP mutations is concordant with earlier clinical descriptions of FHS (Table 1 and Figure 1), and nearly all individuals have short stature and expressive-language impairment. Despite the remarkable similarity among mutations seen in our study subjects, cognitive outcomes ranging from ‘‘normal’’ to ‘‘significant intellectual disability’’ were reported. It is
unclear whether genetic modifier(s) and/or currently unidentified environmental factors are responsible. Many of our study subjects had additional systemic malformations, particularly genitourinary (eight individuals) and cardiac (three individuals) malformations. Again, genotype-phenotype correlations explaining these features are lacking. Given that FHS is a dominant condition exhibiting a high de novo mutation rate, one might expect a paternal age effect to be present, and indeed the mean paternal age of the affected individuals was advanced (36.9 years; range: 29–44 years). Importantly, our findings suggest a basis for the longrecognized phenotypic overlap between FHS and RTS, the latter of which is caused by alterations in CBP or its homolog, p300.14–16 Because alterations in both CBP and SRCAP are expected to produce widespread target-gene dysregulation, further studies are needed before we can determine which transcriptional targets are primarily responsible for each phenotype and whether any of these might be valid therapeutic targets. The availability of a molecular test for FHS will greatly improve the reliability of a formerly challenging clinical diagnosis. Historically, a diagnosis of FHS has relied upon the presence of typical facial features because many other key findings (e.g., short stature and language impairment) are nonspecific. The advent of molecular diagnosis for this condition will allow us to gain a better appreciation of the true prevalence and phenotypic spectrum of FHS. Supplemental Data Supplemental Data include one figure and can be found with this article online at http://www.cell.com/AJHG/.
Acknowledgments The authors would first like to thank the study participants and their families, without whose participation and cooperation this work would not have been possible. This work was funded
312 The American Journal of Human Genetics 90, 308–313, February 10, 2012
by the government of Canada through Genome Canada, the Canadian Institutes of Health Research (CIHR), and the Ontario Genomics Institute (OGI-049). Additional funding was provided by Genome Que´bec and Genome British Columbia. K.M.B. is supported by a Clinical Investigatorship Award from the CIHR Institute of Genetics. This work was selected for study by the FORGE Canada Steering Committee, consisting of K. Boycott (University of Ottawa), J. Friedman (University of British Columbia), J. Michaud (University of Montreal), F. Bernier (University of Calgary), M. Brudno (University of Toronto), B. Fernandez (Memorial University), B. Knoppers (McGill University), M. Samuels (Universite´ de Montreal), and S. Scherer (University of Toronto). Received: November 22, 2011 Revised: December 5, 2011 Accepted: December 7, 2011 Published online: January 19, 2012
5.
6.
7.
8.
Web Resources
9.
The URLs for data presented herein are as follows: Database of Genomic Variants, http://projects.tcag.ca/variation/ FASTX-Toolkit, http://hannonlab.cshl.edu/fastx_toolkit/ NHLBI Exome Variant Server, http://evs.gs.washington.edu/EVS/ Online Mendelian Inheritance in Man (OMIM), http://www. omim.org Picard, http://picard.sourceforge.net/ SAMtools, http://samtools.sourceforge.net/
10.
11.
Accession Numbers The NCBI accession number for the SRCAP sequence reported in this paper is NM_006662.2 and local identifiers are as follows:
NM_006662.2: c.7330C>T (NCBI ss477606270) NM_006662.2: c.7303C>T (NCBI ss477606271) NM_006662.2: c.7549delC (NCBI ss477606272) NM_006662.2: c.7218_7219delTC (NCBI ss477606273) NM_006662.2: c.7316dupC (NCBI ss477606274)
12.
13.
14.
References 1. Pelletier, G., and Feingold, M. (1973). Case report 1. In Syndrome Identification, D. Bergsma, ed. (White Plains, NY: National Foundation-March of Dimes), pp. 8–9. 2. Leisti, J., Hollister, D.W., and Rimoin, D.L. (1975). The FloatingHarbor syndrome. Birth Defects Orig. Artic. Ser. 11, 305. 3. Robinson, P.L., Shohat, M., Winter, R.M., Conte, W.J., Gordon-Nesbitt, D., Feingold, M., Laron, Z., and Rimoin, D.L. (1988). A unique association of short stature, dysmorphic features, and speech impairment (Floating-Harbor syndrome). J. Pediatr. 113, 703–706. 4. White, S.M., Morgan, A., Da Costa, A., Lacombe, D., Knight, S.J., Houlston, R., Whiteford, M.L., Newbury-Ecob,
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